The compositions of the invention relate to the field of proteins which inhibit serine protease activity. The invention also relates to the field of nucleic acid constructs, vectors and host cells for producing serine protease inhibiting proteins, pharmaceutical compositions containing the protein, and methods for their use.
Problem Addressed
Blood loss is a serious complication of major surgeries such as open heart surgery and other complicated procedures. Cardiac surgery patients account for a significant proportion of transfused donor blood. Blood transfusion carries risks of disease transmission and adverse reactions. In addition, donor blood is expensive and demands often exceed supply. Pharmacological methods for reducing blood loss and the resultant need for transfusion have been described (reviewed by Scott et al., Ann. Thorac. Surg. 50: 843-851, 1990).
Protein Serine Protease Inhibitor
Aprotinin, a bovine serine protease inhibitor of the Kunitz family is the active substance in the medicament Trasylol(copyright). Aprotinin (Trasylol(copyright)) has been reported as being effective in reducing perioperative blood loss (Royston et al., Lancet ii: 1289-1291, 1987; Dietrich et al., Thorac. Cardiovasc. Surg. 37: 92-98, 1989; Fraedrich et al., Thorac. Cardiovasc. Surg. 37: 89-91, 1989); W. van Oeveren et al. (1987), Ann Thorac. Surg. 44, pp 640-645; Bistrup et al., (1988) Lancet I, 366-367), but adverse effects, including hypotension and flushing (Bohrer et al., Anesthesia 45: 853-854, 1990) and allergic reactions (Dietrich et al., Supra) have been reported. Use of aprotinin in patients previously exposed to it is not recommended (Dietrich et al., Supra). Trasylol(copyright) has also been used for the treatment of hyperfibrinolytic hemorrhages and traumatic hemorrhagic shock.
Aprotinin is known to inhibit several serine proteases including trypsin, chymotrypsin, plasmin and kallikrein, and is used therapeutically in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction (Trapnell et al., (1974) Brit J. Surg. 61: 177; J. McMichan et al., (1982) Circulatory Shock 9: 107; Auer et al., (1979)Acta Neurochir. 49: 207; Sher (1977) Am J. Obstet. Gynecol. 129: 164; Schneider (1976), Artzneim.-Firsch. 26: 1606). It is generally thought that Trasylol(copyright) reduces blood loss in vivo through inhibition of kallikrein and plasmin. It has been found that aprotinin (3-58, Arg15, Ala17, Ser42) exhibits improved plasma kallikrein inhibitory potency as compared to native aprotinin itself (WO 89/10374).
Problems With Aprotinin
Because aprotinin is of bovine origin, there is a finite risk of inducing anaphylaxis in human patients upon re-exposure to the drug. Thus, a human functional equivalent to aprotinin, by virtue of a lower risk of anaphylaxis, would be most useful and desirable to have.
Aprotinin is also nephrotoxic in rodents and dogs when administered repeatedly at high dose (Bayer, Trasylol(copyright), Inhibitor of proteinase; Glasser et al., in xe2x80x9cVerhandlungen der Deutschen Gesellschaft fur Innere Medizin, 78. Kongressxe2x80x9d, Bergmann, Munchen, 1972 pp. 1612-1614). One hypothesis ascribes this effect to the accumulation of aprotinin in the negatively charged proximal tubules of the kidney, due to its high net positive charge (WO 93/14120).
Accordingly, an object of the present invention is to identify human proteins with functional activity similar to aprotinin. It was also an object of the instant invention to identify human proteins, that would be less charged, yet exhibit the same, highly similar, or improved protease specificities as found for aprotinin, especially with respect to the potency of plasmin and kallikrein inhibition. Such inhibitors could then be used repeatedly as medicaments in human patients with reduced risk of adverse immune response and reduced nephrotoxicity.
The instant invention provides for a purified human serine protease inhibitor which can specifically inhibit kallikrein, that has been isolated from human placental tissue via affinity chromatography.
The instant invention provides a newly identified human protein herein called human placental bikunin that contains two serine protease inhibitor domains of the Kunitz class. In one particular embodiment, the instant invention embodies a protein having the amino acid sequence:
In a prefered embodiment the instant invention provides for native human placental bikunin protein having the amino acid sequence:
In one aspect, the biological activity of the protein of the instant invention is that it can bind to and substantially inhibit the biological activity of trypsin, human plasma and tissue kallikreins, human plasmin and Factor XIIa. In a preferred embodiment, the present invention provides for a native human placental bikunin protein, in glycosylated form. In a further embodiment the instant invention encompasses native human bikunin protein which has been formed such that it contains at least one cysteine-cysteine disulfide bond. In a preferred embodiment, the protein contains at least one intra-chain cysteine-cysteine disulfide bond formed between a pair of cysteines selected from the group consisting of CYS11-CYS61, CYS20-CYS44, CYS36-CYS57, CYS106-CYS156, CYS115-CYS139, and CYS131-CYS152, wherein the cysteines are numbered according to the amino acid sequence of native human placental bikunin. One of ordinary skill will recognize that the protein of the instant invention may fold into the proper three-dimensional conformation, such that the biological activity of native human bikunin is maintained, where none, one or more, or all of the native intra-chain cysteine-cysteine disulfide bonds are present. In a most preferred embodiment, the protein of the instant invention is properly folded and is formed with all of the proper native cysteine-cysteine disulfide bonds.
Active protein of the instant invention can be obtained by purification from human tissue, such as placenta, or via synthetic protein chemistry techniques, as illustrated by the Examples below. It is also understood that the protein of the instant invention may be obtained using molecular biology techniques, where self-replicating vectors are capable of expressing the protein of the instant invention from transformed cells. Such protein can be made as non-secreted, or secreted forms from transformed cells. In order to facilitate secretion from transformed cells, to enhance the functional stability of the translated protein, or to aid folding of the bikunin protein, certain signal peptide sequences may be added to the NH2-terminal portion of the native human bikunin protein.
In one embodiment, the instant invention thus provides for the native human bikunin protein with at least a portion of the native signal peptide sequence intact. Thus one embodiment of the invention provides for native human bikunin with at least part of the signal peptide, having the amino acid sequence:
In a prefered embodiment the instant invention provides for a native human placental bikunin protein with part of the leader sequence intact, having the amino acid sequence of SEQ ID NO: 52 with an intact leader segment having the amino acid sequence:
MAQLCGL RRSRAFLALL GSLLLSGVLAxe2x88x921 (SEQ ID NO: 53)
In another embodiment, the instant invention provides for bikunin protein with part of the leader sequence intact, having the amino acid sequence of SEQ ID NO: 52 with the intact leader segment having the amino acid sequence:
MLR AEADGVSRLL GSLLLSGVLAxe2x88x921 (SEQ ID NO: 54)
In a preferred numbering system used herein the amino acid numbered+1 is assigned to the NH2-terminus of the amino acid sequence for native human placental bikunin. One will readily recognize that functional protein fragments can be derived from native human placental bikunin, which will maintain at least part of the biological activity of native human placental bikunin, and act as serine protease inhibitors.
In one embodiment, the protein of the instant invention comprises a fragment of native human placental bikunin, which contains at least one functional Kunitz-like domain, having the amino acid sequence of native human placental bikunin amino acids 7-159, hereinafter called xe2x80x9cbikunin (7-159)xe2x80x9d. Thus the instant invention embodies a protein having the amino acid sequence:
where the amino acid numbering corresponds to that of the amino acid sequence of native human placental bikunin. Another functional variant of this embodiment can be the fragment of native human placental bikunin, which contains at least one functional Kunitz-like domain, having the amino acid sequence of native human placental bikunin amino acids 11-156, bikunin (11-156)
One can recognize that the individual Kunitz-like domains are also fragments of the native placental bikunin. In particular, the instant invention provides for a protein having the amino acid sequence of a first Kunitz-like domain consisting of the amino acid sequence of native human placental bikunin amino acids 7-64, hereinafter called xe2x80x9cbikunin (7-64)xe2x80x9d. Thus in one embodiment the instant invention encompasses a protein which contains at least one Kunitz-like domain having the amino acid sequence:
where the amino acid numbering corresponds to that of the amino acid sequence of native human placental bikunin. Another form of the protein of the instant invention can be a first Kunitz-like domain consisting of the amino acid sequence of native human placental bikunin amino acids 11-61, xe2x80x9cbikunin (11-61)xe2x80x9d having the amino acid sequence:
The instant invention also provides for a protein having the amino acid sequence of a Kunitz-like domain consisting of the amino acid sequence of native human placental bikunin amino acids 102-159, hereinafter called xe2x80x9cbikunin (102-159)xe2x80x9d. Thus one embodiment the instant invention encompasses a protein which contains at least one Kunitz-like domain having the amino acid sequence:
where the amino acid numbering corresponds to that of the amino acid sequence of native human placental bikunin. Another form of this domain can be a Kunitz-like domain consisting of the amino acid sequence of native human placental bikunin amino acids 106-156, xe2x80x9cbikunin (106-156)xe2x80x9d having the amino acid sequence:
Thus one of ordinary skill will recognize that fragments of the native human bikunin protein can be made which will retain at least some of the native protein biological activity. Such fragments can also be combined in different orientations or multiple combinations to provide for alternative proteins which retain some of, the same, or more biological activity of the native human bikunin protein.
One will readily recognize that biologically active protein of the instant invention may comprise one or more of the instant Kunitz-like domains in combination with additional Kunitz-like domains from other sources. Biologically active protein of the instant invention may comprise one or more of the instant Kunitz-like domains in combination with additional protein domains from other sources with a variety of biological activities. The biological activity of the protein of the instant invention can be combined with that of other known protein or proteins to provide for multifunctional fusion proteins having predictable biological activity. Thus, in one embodiment, the instant invention encompasses a protein which contains at least one amino acid sequence segment the same as, or functionally equivalent to the amino acid sequence of either SEQ ID NO: 5 or SEQ ID NO: 7.
An open reading frame which terminates at an early stop codon can still code for a functional protein. The instant invention encompasses such alternative termination, and in one embodiment provides for a protein of the amino acid sequence:
In one embodiment, the instant invention provides for substantially purified, or recombinantly produced native human bikunin protein with an intact segment of the leader sequence, and at least a portion of the native transmembrane region intact. Thus one embodiment of the invention provides for native human bikunin, with an intact leader sequence, and with at least part of the transmembrane domain (underlined), having an amino acid sequence selected from:
where sequence 1) is EST derived consensus SEQ ID NO: 45, 2) is PCR clone SEQ ID NO:47, and 3) is lambda cDNA clone SEQ ID NO:49. In a preferred embodiment a protein of the instant invention comprises one of the amino acid sequence of SEQ ID NO: 45, 47 or 49 wherein the protein has been cleaved in the region between the end of the last Kunitz domain and the transmembrane region.
The instant invention also embodies the protein wherein the signal peptide is deleted. Thus the instant invention provides for a protein having the amino acid sequence of SEQ ID NO: 52 continuous with a transmembrane amino acid sequence:
a transmembrane amino acid sequence:
or a transmembrane amino acid sequence:
The protein amino acid sequences of the instant invention clearly teach one of the art the appropriate nucleic acid sequences which can be used in molecular biology techniques to produce the proteins of the instant invention. Thus, one embodiment of the instant invention provides for a nucleic acid sequence which encodes for a human bikunin having the consensus DNA sequence of FIG. 3 (SEQ ID NO: 9), which translates into the amino acid sequence for native human placental bikunin sequence of FIG. 3 (SEQ ID NO: 10). In another embodiment, the instant invention provides for a consensus nucleic acid sequence of FIG. 4C (SEQ ID NO: 51) which encodes for an amino acid sequence of FIG. 4D (SEQ ID NO: 45).
In a preferred embodiment, the instant invention provides for a nucleic acid sequence which encodes for native human placental bikunin having the DNA sequence of FIG. 4F (SEQ ID NO: 48) which encodes for the protein sequence of SEQ ID NO: 49. In an another embodiment, the instant invention provides for a nucleic acid sequence of FIG. 4E (SEQ ID NO: 46) which encodes for a protein sequence of SEQ ID NO: 47.
One can easily recognize that certain allelic mutations, and conservative substitutions made in the nucleic acid sequence can be made which will still result in a protein amino acid sequence encompassed by the instant invention. One of skill in the art can recognize that certain natural allelic mutations of the protein of the instant invention, and conservative substitutions of amino acids in the protein of the instant invention will not significantly alter the biological activity of the protein, and are encompassed by the instant invention.
The instant invention also provides for pharmaceutical compositions containing human placental bikunin and fragments thereof that are useful for the reduction of perioperative blood loss in a patient undergoing surgery.
The present invention also provides methods for reducing perioperative blood loss in a patient undergoing surgery, wherein an effective amount of the disclosed human serine protease inhibitors of the present invention in a biologically compatible vehicle is administered to the patient.
The present invention also provides for variants of placental bikunin, and the specific Kunitz domains described above, that contain amino acid substitutions that alter the protease specificity. Preferred sites of substitution are indicated below as positions Xaa1 through Xaa32 in the amino acid sequence for native placental bikunin. Substitutions at Xaa1 through Xaa16 are also preferred for variants of bikunin (7-64), while substitutions at Xaa17 through Xaa32 are preferred for variants of bikunin (102-159).
Thus the present invention embodies protein having an amino acid sequence:
where Xaa1-Xaa32 each independently represents a naturally occurring amino acid residue except Cys, with the proviso that at least one of the amino acid residues Xaa1-Xaa32 is different from the corresponding amino acid residue of the native sequence.
In the present context, the term xe2x80x9cnaturally occurring amino acid residuexe2x80x9d is intended to indicate any one of the 20 commonly occurring amino acids, i.e., Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.
By substituting one or more amino acids in one or more of the positions indicated above, it may be possible to change the inhibitor specificity profile of native placental bikunin or that of the individual Kunitz-like domains, bikunin(7-64) or bikunin (102-159) so that it preferentially inhibits other serine proteases such as, but not limited to, the enzymes of the complement cascade, TF/FVIIa, FXa, thrombin, neutrophil elastase, cathepsin G or proteinase-3.
Examples of preferred variants of placental bikunin include those wherein Xaa1 is an amino acid residue selected from the group consisting of His, Glu, Pro, Ala, Val or Lys, in particular wherein Xaa1 is His or Pro; or wherein Xaa2 is an amino acid residue selected from the group consisting of Val, Thr, Asp, Pro, Arg, Tyr, Glu, Ala, Lys, in particular wherein Xaa2 is Val or Thr; or wherein Xaa3 is an amino acid residue selected from the group consisting of Arg, Pro, Ile, Leu, Thr, in particular wherein Xaa3 is Arg or Pro; or wherein Xaa4 is an amino acid residue selected from the group consisting of Arg, Lys and Ser, Gln, in particular wherein Xaa4 is Arg or Lys; or wherein Xaa5 is an amino acid residue selected from the group consisting of Ala, Gly, Asp, Thr, in particular wherein Xaa5 is Ala; or wherein Xaa6 is an amino acid residue selected from the group consisting of Ser, Ile, Tyr, Asn, Leu, Val, Arg, Phe, in particular wherein Xaa6 is Ser or Arg; or wherein Xaa7 is an amino acid residue selected from the group consisting of Met, Phe, Ile, Glu, Leu, Thr and Val, in particular wherein Xaa7 is Met or Ile; or wherein Xaa8 is an amino acid residue selected from the group consisting of Pro, Lys, Thr, Gln, Asn, Leu, Ser or Ile, in particular wherein Xaa8 is Pro or Ile; or wherein Xaa9 is an amino acid residue selected from the group consisting of Arg, Lys or Leu, in particular wherein Xaa9 is Arg: or wherein Xaa10 is an amino acid residue selected from the group consisting of Val, Ile, Lys, Ala, Pro, Phe, Trp, Gln, Leu and Thr, in particular wherein Xaa10 is Val; or wherein Xaa11 is an amino acid residue selected from the group consisting of Gly, Ser and Thr, in particular wherein Xaa11 is Gly; or wherein Xaa12 is an amino acid residue selected from the group consisting of Asp, Arg, Glu, Leu, Gln, Gly, in particular wherein Xaa12 is Asp or Arg; or wherein Xaa13 is an amino acid residue selected from the group consisting of Gly and Ala; or wherein Xaa14 is an amino acid residue selected from the group consisting of Asn or Lys; or wherein Xaa15  is an amino acid residue selected from the group consisting of Gly, Asp, Leu, Arg, Glu, Thr, Tyr, Val, and Lys, in particular wherein Xaa15 is Leu or Lys; or wherein Xaa16 is an amino acid residue selected from the group consisting of Val, Gln, Asp, Gly, Ile, Ala, Met, and Val, in particular wherein Xaa16 is Val or Ala; or wherein Xaa17 is an amino acid residue selected from the group consisting of His, Glu, Pro, Ala, Lys and Val, in particular wherein Xaa17 is Glu or Pro; or wherein Xaa18 is an amino acid residue selected from the group consisting of Val, Thr, Asp, Pro, Arg, Tyr, Glu, Ala or Lys, in particular wherein Xaa18 is Thr; or wherein Xaa19 is an amino acid residue selected from the group consisting of Arg, Pro, Ile, Leu or Thr, in particular wherein Xaa19 is Pro; or wherein Xaa20 is an amino acid residue selected from the group consisting of Arg, Lys, Gln and Ser, in particular wherein Xaa20 is Arg or Lys; or wherein Xaa21 is an amino acid residue selected from the group consisting of Ala, Asp, Thr or Gly; in particular wherein Xaa21 is Ala; or wherein Xaa22 is an amino acid residue selected from the group consisting of Ser, Ile, Tyr, Asn, Leu, Val, Arg or Phe, in particular wherein Xaa22 is Ser or Arg; or wherein Xaa23 is an amino acid residue selected from the group consisting of Met, Phe, Ile, Glu, Leu, Thr and Val, in particular wherein Xaa23 is Phe or Ile; or wherein Xaa24 is an amino acid residue selected from the group consisting of Pro, Lys, Thr, Asn, Leu, Gln, Ser or Ile, in particular wherein Xaa24 is Pro or Ile; or wherein Xaa25 is an amino acid residue selected from the group consisting of Arg, Lys or Leu, in particular wherein Xaa25 is Arg: or wherein Xaa26 is an amino acid residue selected from the group consisting of Val, Ile, Lys, Leu, Ala, Pro, Phe, Gln, Trp and Thr, in particular wherein Xaa26 is Val or Ile; or wherein Xaa27 is an amino acid residue selected from the group consisting of Gly, Ser and Thr, in particular wherein Xaa27 is Gly; or wherein Xaa28 is an amino acid residue selected from the group consisting of Asp, Arg, Glu, Leu, Gly or Gln, in particular wherein Xaa28 is Arg; or wherein Xaa29 is an amino acid residue selected from the group consisting of Gly and Ala; or wherein Xaa30 is an amino acid residue selected from the group consisting of Asn or Lys; or wherein Xaa31 is an amino acid residue selected from the group consisting of Gly, Asp, Leu, Arg, Glu, Thr, Tyr, Val, and Lys, in particular wherein Xaa31 is Arg or Lys; or wherein Xaa32 is an amino acid residue selected from the group consisting of Val, Gln, Asp, Gly, Ile, Ala, Met, and Thr, in particular wherein Xaa32 is Gln or Ala.